Showing papers in "Planta in 1999"
TL;DR: In this paper, a modified version of the TBARS method was proposed for assessing the presence of malondialdehyde (MDA) and other non-MDA compounds absorbing at 532 nm.
Abstract: The occurrence of malondialdehyde (MDA), a secondary end product of the oxidation of polyunsatu- rated fatty acids, is considered a useful index of general lipid peroxidation A common method for measuring MDA, referred to as the thiobarbituric acid-reactive- substances (TBARS) assay, is to react it with thiobar- bituric acid (TBA) and record the absorbance at 532 nm However, many plants contain interfering compounds that also absorb at 532 nm, leading to overestimation of MDA values Extracts of plant tissues including purple eggplant (Solanum melongena L) fruit, carrot (Daucus carota L) roots, and spinach (Spinacia oleracea L) leaves were assessed for the presence of MDA and other non-MDA compounds absorbing at 532 nm A method described herein corrects for these interferences by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA The reliability and eAciency of this spectrophotometric method was assessed by altering the relative ratios of exogenous MDA additions and/or extracts of red cabbage (Brassica oleracea L) leaves containing inter- fering compounds and then measuring MDA recovery Reliability was also validated through high-performance liquid chromatography and high-performance liquid chromatography-mass spectrometry techniques Results indicated that over 90% of exogenously added MDA could be recovered through the improved protocol If there were no corrections for interfering compounds, MDA equivalents were overestimated by up to 965% Interfering compounds were not detected in vegetables such as lettuce (Lactuca sativa L) and spinach which had low or negligible concentrations of anthocyanidin derivatives Comparisons between the TBARS method presented here and two currently accepted protocols indicated that the new modified method exhibits greater accuracy for quantifying TBA-MDA levels in tissues containing anthocyanins and/or other interfering com- pounds This modified protocol represents a facile and rapid method for assessment of lipid peroxidation in virtually all plant species that contain interfering com- pounds
3,090 citations
TL;DR: Salicylic acid (SA) pre-treatment has been shown to decrease net photosynthesis, stomatal conductivity and transpiration at the growth temperature (22/20 °C).
Abstract: The addition of 0.5 mM salicylic acid (SA) to the hydroponic growth solution of young maize (Zea mays L.) plants under normal growth conditions provided protection against subsequent low-temperature stress. This observation was confirmed by chlorophyll fluorescence parameters and electrolyte leakage measurements. In addition, 1 d of 0.5 mM SA pre-treatment decreased net photosynthesis, stomatal conductivity and transpiration at the growth temperature (22/20 °C). Since there was only a slight decrease in the ratio of variable to maximal fluorescence (Fv/Fm) the decrease in photosynthetic activity is not due to a depression in photosystem II. The analysis of antioxidant enzymes showed that whereas SA treatment did not cause any change in ascorbate peroxidase (EC 1.11.1.11) and superoxide dismutase (EC 1.15.1.1) activities, there was a decrease in catalase (EC 1.11.1.6) activity, and an increase in guaiacol peroxidase (EC 1.11.1.7) and glutathione reductase (EC 1.6.4.2) activities after the 1-d SA treatment at 22/20 °C. In native polyacrylamide gels there was, among the peroxidase isoenzymes, a band which could be seen only in SA-treated plants. It is suggested that the pre-treatment of maize plants with SA at normal growth temperature may induce antioxidant enzymes which lead to increased chilling tolerance.
555 citations
TL;DR: It is demonstrated that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency, suggesting the induction of a high-affinity Pi-uptake system.
Abstract: Release of large amounts of citric acid from specialized root clusters (proteoid roots) of phosphorus (P)-deficient white lupin (Lupinus albus L.) is an efficient strategy for chemical mobilization of sparingly available P sources in the rhizosphere. The present study demonstrates that increased accumulation and exudation of citric acid and a concomitant release of protons were predominantly restricted to mature root clusters in the later stages of P deficiency. Inhibition of citrate exudation by exogenous application of anion-channel blockers such as ethacrynic- and anthracene-9-carboxylic acids may indicate involvement of an anion channel. Phosphorus-deficiency-induced accumulation and subsequent exudation of citric acid seem to be a consequence of both increased biosynthesis and reduced metabolization of citric acid in the proteoid root tissue, indicated by increased in-vitro activity and enzyme protein levels of phosphoenolpyruvate carboxylase (EC 4.1.1.31), and reduced activity of aconitase (EC 4.2.1.3) and root respiration. Similar to citric acid, acid phosphatase, which is secreted by roots and involved in the mobilization of the organic soil P fraction, was released predominantly from proteoid roots of P-deficient plants. Also 33Pi uptake per unit root fresh-weight was increased by approximately 50% in juvenile and mature proteoid root clusters compared to apical segments of non-proteoid roots. Kinetic studies revealed a K
m of 30.7 μM for Pi uptake of non-proteoid root apices in P-sufficient plants, versus K
m values of 8.5–8.6 μM for non-proteoid and juvenile proteoid roots under P-deficient conditions, suggesting the induction of a high-affinity Pi-uptake system. Obviously, P-deficiency-induced adaptations of white lupin, involved in P acquisition and mobilization of sparingly available P sources, are predominantly confined to proteoid roots, and moreover to distinct stages during proteoid root development.
424 citations
TL;DR: It is suggested that, acting as an antioxidant, NO can strongly counteract many ROS-mediated cytotoxic processes in plants and the evidence of NO functionality in the plant kingdom is strengthened by this work.
Abstract: Many environmental conditions subject plants to oxidative stress, in which reactive oxygen species (ROS) are overproduced. These ROS act as transduction signals in plant defense responses, but also cause effects that result in cellular damage. Since nitric oxide (NO) is a bioactive molecule able to scavenge ROS, we analyzed its effect on some cytotoxic processes produced by ROS in potato (Solanum tuberosum L. cv. Pampeana) leaves. Two NO donors: (i) sodium nitroprusside and (ii) a mixed solution of ascorbic acid and NaNO2, were able to prevent chlorophyll loss mediated by the methyl viologen herbicide diquat (a ROS generator), with effective concentrations falling between 10 and 100 μM of the donors. This protection was mimicked by thiourea and penicillamine, two antioxidant compounds. Residual products from NO generation and decomposition failed to prevent chlorophyll decline. A specific NO scavenger, the potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), arrested NO-mediated chlorophyll protection. In addition, some events mediated by ROS during infection of potato leaves with Phytophthora infestans (race 1, 4, 7, 8, 10, 11, mating type A2) were also examined. In this sense, NO proved to markedly decrease ion leakage and the number of lesions, indicative of cell death, produced upon infection in potato leaves. The NO-mediated decrease in ion leakage was also inhibited by carboxy-PTIO. Fragmentation of DNA diminished when P. infestans-infected potato leaves were treated with 100 μM SNP. These results suggest that, acting as an antioxidant, NO can strongly counteract many ROS-mediated cytotoxic processes in plants. Moreover, the evidence of NO functionality in the plant kingdom is strengthened by this work.
300 citations
TL;DR: In the following century, functional aspects of secondary metabolism were rejected or ignored by most plant scientists and those discussions of function that did appear were met with controversy (for details see Mothes 1955; Swain 1977; Haslam 1986; Luckner 1990).
Abstract: In his textbook published 125 years ago, Julius Sachs, one of the pioneers of plant physiology, made a clear distinction between products of secondary metabolism ('Nebenprodukte des Stoffwechsels') and those of pri mary metabolism1. The definition is still valid but Sachs did not refer to any functions of secondary metabolism. This, however, was done by his contemporaries such as Ernst Stahl (1888) a former student of Sachs, the Austrian physician and botanist Anton Kerner von Marilaun (1890) and the prominent Belgian plant scientist Leo Errera (1886), all of whom emphasized the importance of plant chemicals as protectives against herbivores. In the following century, functional aspects of secondary metabolism were rejected or ignored by most plant scientists and those discussions of function that did appear were met with controversy (for details see Mothes 1955; Swain 1977; Haslam 1986; Luckner 1990). Curiously, it was entomologists who revived the early functional view of
289 citations
TL;DR: It was concluded that the conductance of membranes of endodermal and stelar cells may be responsible for the observed diurnal rhythm in root Lpr and when mRNAs from roots were probed with cDNA from the Arabidopsis aquaporin AthPIP1a gene, an abundant transcript was found to vary in abundance diurnally under high-stringency conditions.
Abstract: The hydraulic conductivity of excised roots (Lpr) of the legume Lotus japonicus (Regel) K. Larsen grown in mist (aeroponic) and sand cultures, was found to vary over a 5-fold range during a day/night cycle. This behaviour was seen when Lpr was measured in roots exuding, either under root pressure (osmotic driving force), or under an applied hydrostatic pressure of 0.4 MPa which produced a rate of water flow similar to that in a transpiring plant. A similar daily pattern of variation was seen in plants grown in natural daylight or in controlled-environment rooms, in plants transpiring at ambient rates or at greatly reduced rates, and in plants grown in either aeroponic or sand culture. When detached root systems were connected to a root pressure probe, a marked diurnal variation was seen in the root pressure generated. After excision, this circadian rhythm continued for some days. The hydraulic conductivity of the plasma membrane of individual root cells was measured during the diurnal cycle using a cell pressure probe. Measurements were made on the first four cell layers of the cortex, but no evidence of any diurnal fluctuation could be found. It was concluded that the conductance of membranes of endodermal and stelar cells may be responsible for the observed diurnal rhythm in root Lpr. When mRNAs from roots were probed with cDNA from the Arabidopsis aquaporin AthPIP1a gene, an abundant transcript was found to vary in abundance diurnally under high-stringency conditions. The pattern of fluctuations resembled closely the diurnal pattern of variation in root Lpr. The plasma membranes of root cells were found to contain an abundant hydrophobic protein with a molecular weight of about 31 kDa which cross-reacted strongly to an antibody raised against the evolutionarily conserved N-terminal amino acid sequence of AthPIP1a.
264 citations
TL;DR: While the mechanisms of many plant-herbivore interactions are not well understood, the ecological complexity of plant responses against herbivores is well recognized, as the defense response elicited by volicitin underscores.
Abstract: Plants must fend off a myriad of aggressive biotic agents and this biotic onslaught occurs across the complete spectrum of spatial scales from pathogens that attack single cells (Somssich and Hahlbrock 1998) to herbi vores that select their hosts by characteristics of the community in which a plant grows (Hay 1986). The recent advances in understanding plant defense respons es that function on the cellular level against pathogens have been awe-inspiring, particularly with regard to the mechanisms of pathogen recognition (Baker et al. 1997). In contrast, our understanding of how plants recognize attack from herbivores is in its infancy; the first herbivore-specific elicitor (volicitin) was only just re cently characterized (Alborn et al. 1997). While the mechanisms of many plant-herbivore interactions are not well understood, the ecological complexity of plant responses against herbivores is well recognized, as the defense response elicited by volicitin underscores. Vol icitin [N-(17-hydroxylinolenoyl)-L-glutamine], isolated from the oral secretions of beet armyworm (Spodoptera exigua) larvae, increases the emission of volatile organic compounds when applied to corn (Alborn et al. 1997). How these volatile emissions function as a defense highlights a key difference between plant defenses against pathogens and herbivores: the physiological autonomy of herbivores, which allow plants to utilize components of their ecological community for defense.
255 citations
TL;DR: In inoculating axenic saltmarsh bulrush plants with bacteria isolated from the rhizosphere of plants collected from the field, these plants accumulated significantly more Se and Hg compared to axenic controls.
Abstract: The role of rhizosphere bacteria in facilitating Se and Hg accumulation in two wetland plants, saltmarsh bulrush (Scirpus robustus Pursh) and rabbitfoot grass (Polypogon monspeliensis (L.) Desf.), was studied. Ampicillin-amended plants (i.e., with inhibited rhizosphere bacteria) supplied with Na2SeO4 or HgCl2 had significantly lower concentrations of Se and Hg, respectively, in roots than plants without ampicillin. These results were confirmed by inoculating axenic saltmarsh bulrush plants with bacteria isolated from the rhizosphere of plants collected from the field; these plants accumulated significantly more Se and Hg compared to axenic controls. Therefore, rhizosphere bacteria can increase the efficiency of Se and Hg phytoremediation by promoting the accumulation of Se and Hg in tissues of wetland plants.
203 citations
TL;DR: It is suggested that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices.
Abstract: The diaminobenzidine (DAB) staining technique was used to examine the accumulation of H2O2 in parts of roots of Medicago truncatula Gaertn. colonized by the arbuscular mycorrhiza (AM)-forming fungus Glomus intraradices Schenk and Smith. At the cellular level, the combination of bright-field and fluorescence microscopy revealed that a brownish stain, indicative of H2O2 accumulation was present within cortical root cells in the space occupied by arbuscules. Accumulation of H2O2 was especially pronounced in cells containing arbuscules that were clumped and less branched. Moreover, H2O2 accumulated around hyphal tips attempting to penetrate a host cell. In contrast, no H2O2 accumulation was observed in hyphal tips growing along the middle lamella, or in appressoria or vesicles. On the basis of these findings we suggest that a locally restricted oxidative burst is involved in the temporal and spatial control of the intracellular colonization of M. truncatula cells by the AM-forming fungus G. intraradices.
190 citations
TL;DR: The results confirm the general belief that dehydrins serve desiccation tolerance, and suggest that a major function is to rescue the metabolic processes that are required for survival and re-growth.
Abstract: Dehydrins accumulate in various plant tissues during dehydration. Their physiological role is not well understood, but it is commonly assumed that they assist cells in tolerating dehydration. Since in perennials the ability of the shoot apex to withstand dehydration is pivotal for survival through winter, we investigated if and how dehydrins may be involved. A first step in assessing such a role is the identification of their subcellular location. We therefore mapped the location of dehydrin homologues, abscisic acid-responsive (RAB 16-like) polypeptides, in the apex of birch (Betula pubescens Ehrh.). In non-cold-acclimated plants a single low-abundant RAB 16-member (a 33-kDa polypeptide) was produced, and localized in the cytoplasm only. During cold acclimation two additional members were produced (24 and 30 kDa) and accumulated in nuclei, storage protein bodies and starch-rich amyloplasts. Western blots of proteins isolated from purified starch granules and from protein bodies revealed the presence of the 24-kDa dehydrin. Since starch and protein reserves are gradually consumed during winter, serving cell maintenance, starch- and protein-degrading enzymes must remain locally active. We therefore investigated the hypothesis that dehydrins might create local pools of water in otherwise dehydrated cells, thereby maintaining enzyme function. In agreement with our hypothesis, enzyme assays showed that under conditions of low water activity a partially purified dehydrin fraction was able to improve the activity of α-amylase (EC 3.2.1.1.) relative to fractions from which dehydrin was removed by immunoprecipitation. The results confirm the general belief that dehydrins serve desiccation tolerance, and suggest that a major function is to rescue the metabolic processes that are required for survival and re-growth.
168 citations
TL;DR: Results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems.
Abstract: In order to test the possibility of enhancing the production of pharmaceutically valuable scopolamine in transgenic cultures, the 35S-h6h transgene that codes for the enzyme hyoscyamine-6β-hydroxylase (EC 1.14.11.11) was introduced into Hyoscyamus muticus L. strain Cairo (Egyptian henbane). This plant was chosen for its capability to produce very high amounts of tropane alkaloids (up to 6% of the dry weight in the leaves of mature plant). To our knowledge, this is the first time such a large population of transgenic cultures has been studied at the morphological, chemical and genetic levels. A great variation was observed in the tropane alkaloid production among the 43 positive transformants. The best clone (KB7) produced 17 mg/l scopolamine, which is over 100 times more than the control clones. However, conversion of hyoscyamine to scopolamine was still incomplete. The expression of h6h was found to be proportional to the scopolamine production, and was the main reason behind the variation in the scopolamine/hyoscyamine ratio in the hairy-root clones. These results indicate that H. muticus strain Cairo has a potential for even more enhanced scopolamine production with more efficient gene-expression systems.
TL;DR: It is shown that both genes are induced by very low levels of nitrate (50 μM KNO3), however, stronger induction was observed with NRT2:1At than with Nrt1:1 at, probably reflecting the de-repression of the high-affinity transport system during N-starvation.
Abstract: We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO3). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation.
TL;DR: The results suggest that the efficiency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis, and that pine transcripts are correctly processed by the angiosperm translational machinery and that GS1 subunits are assembled in functional holoenzymes.
Abstract: The assimilation of ammonium into organic nitrogen catalyzed by the enzyme glutamine synthetase (GS; EC 6.3.1.2) has been suggested to be the limiting step for plant nitrogen utilization (H-M. Lam et al. 1995, Plant Cell 7: 887-898). We have developed a molecular approach to increase glutamine production in transgenic poplar by the overexpression of a conifer GS gene. A chimeric construct consisting of the cauliflower mosaic virus 35S promoter fused to pine cytosolic GS cDNA and nopaline synthetase polyadenylation region was transferred into pBin19 for transformation of a hybrid poplar clone (INRA 7171-B4, Populus tre- mula · P. alba) via Agrobacterium tumefaciens. Trans- formed poplar lines were selected by their ability to grow on selective medium containing kanamycin. The pres- ence of the introduced gene in the poplar genome was verified by Southern blotting and polymerase chain reaction analysis. Transgene expression was detected in all selected poplar lines at the mRNA level. The detection of the corresponding polypeptide (41 kDa) and increased GS activity in the transgenics suggest that pine transcripts are correctly processed by the angio- sperm translational machinery and that GS1 subunits are assembled in functional holoenzymes. Expression of the pine GS1 gene in poplar was associated with an increase in the levels of total soluble protein and an increase in chlorophyll content in leaves of transformed trees. Furthermore, the mean net growth in height of GS-overexpressing clones was significantly greater than that of non-transformed controls, ranging from a 76% increase in height at 2 months to a 21.3% increase at 6 months. Our results suggest that the eAciency of nitrogen utilization may be engineered in trees by genetic manipulation of glutamine biosynthesis.
TL;DR: Mastoparan interferes with the PIP2-regulated profilin-actin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs.
Abstract: The continuously changing polar cytoplasmic organization during initiation and tip growth of root hairs is reflected by a dynamic redistribution of cytoskeletal elements. The small G-actin binding protein, profilin, which is known to be a widely expressed, potent regulator of actin dynamics, was specifically localized at the tip of root hairs and co-distributed with a diffusely fluorescing apical cap of actin, but not with subapical actin microfilament (MF) bundles. Profilin and actin caps were present exclusively in the bulge of outgrowing root hairs and at the apex of elongating root hairs; both disappeared when tip growth terminated, indicating a tip-growth mechanism that involves profilin-actin interactions for the delivery and localized exocytosis of secretory vesicles. Phosphatidylinositol-4,5-bisphosphate (PIP(2)), a ligand of profilin, was localized almost exclusively in the bulge and, subsequently, formed a weak tip-to-base gradient in the elongating root hairs. When tip growth was eliminated by the MF-disrupting inhibitor cytochalasin D, the apical profilin and the actin fluorescence were lost. Mastoparan, which is known to affect the PIP(2) cycle, probably by stimulating phospholipases, caused the formation of a meshwork of distinct actin MFs replacing the diffuse apical actin cap and, concomittantly, tip growth stopped. This suggests that mastoparan interferes with the PIP(2)-regulated profilin-actin interactions and hence disturbs conditions indispensable for the maintenance of tip growth in root hairs.
TL;DR: It is observed that the presence of truncated sequences of transgenes capable of generating incomplete transcripts, resulting in aberrant RNA species, may be responsible for silencing in transgenic rice lines generated through particle-bombardment-mediated transformation.
Abstract: Transgenic rice (Oryza sativa L.) lines were generated through particle-bombardment-mediated transformation. Hygromycin phosphotransferase (hpt) was used as a selectable marker gene on co-integrate plasmids containing either one or two unselected genes, the Bialaphos-resistance gene (bar) coding for phosphinothricin acetyltransferase and the β-glucuronidase gene (gusA), respectively. Transformants were analyzed to determine possible correlation between expression, integrated transgene copy number and/or complexity of integration patterns. We observed that an increase in transgene copy number did not always lead to a concomitant decrease in expression levels or to silencing through co-suppression. Transgenic lines with four to five copies of integrated transgenes expressed the protein product of both unselected genes stably and at levels comparable to transformants with one or two copies. In the majority of lines we analyzed, expression patterns of the two unselected genes were similar. In lines where transgene silencing was observed, this was independent of position effects. In specific cases, silenced transgenes could be reactivated by treatment with 5-azacytidine, suggesting methylation of cytosine residues. We report that methylation of cytosines may not spread to adjacent regions, hence other transgenes in the vicinity of the silenced transgene remain active. By comparing the structure of transgenic loci with expression patterns of introduced genes, we concluded that the integrity of integrated transgenes was a major factor in the onset of silencing. We observed that the presence of truncated sequences of transgenes capable of generating incomplete transcripts, resulting in aberrant RNA species, may be responsible for silencing.
TL;DR: It is concluded that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation.
Abstract: Jasmonic acid (JA) and salicylic acid (SA) have both been implicated as important signal molecules mediating induced defenses of Nicotiana tabacum L. against herbivores and pathogens. Since the application of SA to a wound site can inhibit both wound-induced JA and a defense response that it elicits, namely nicotine production, we determined if tobacco mosaic virus (TMV) inoculation, with its associated endogenous systemic increase in SA, reduces a plant's ability to increase JA and nicotine levels in response to mechanical damage, and evaluated the consequences of these interactions for the amount of tissue removed by a nicotine-tolerant herbivore, Manduca sexta. Additionally, we determined whether the release of volatile methyl salicylic acid (MeSA) from inoculated plants can reduce wound-induced JA and nicotine responses in uninoculated plants sharing the same chamber. The TMV-inoculated plants, though capable of inducing nicotine normally in response to methyl jasmonate applications, had attenuated wound-induced JA and nicotine responses. Moreover, larvae consumed 1.7- to 2.7-times more leaf tissue from TMV-inoculated plants than from mock-inoculated plants. Uninoculated plants growing in chambers downwind of either TMV-inoculated plants or vials releasing MeSA at 83- to 643-times the amount TMV-inoculated plants release, exhibited normal wound-induced responses. We conclude that tobacco plants, when inoculated with TMV, are unable to elicit normal wound responses, due likely to the inhibition of JA production by the systemic increase in SA induced by virus-inoculation. The release of volatile MeSA from inoculated plants is not sufficient to influence the wound-induced responses of neighboring plants.
TL;DR: The results suggest that the level of free OAS mediates sulfur- and nitrogen-regulation of soybean seed storage-protein composition, similar to that of gene expression driven by the β-subunit promoter.
Abstract: The composition of seed storage proteins is regulated by sulfur and nitrogen supplies. Under conditions of a low sulfur-to-nitrogen ratio, accumulation of the β-subunit of β-conglycinin, a sulfur-poor seed storage protein of soybean (Glycine max [L.] Merr.), is elevated, whereas that of glycinin, a sulfur-rich storage protein, is reduced. Using transgenic Arabidopsis thaliana [L.] Heynh., it was found that the promoter from the gene encoding the β-subunit of β-conglycinin up-regulates gene expression under sulfur deficiency and down-regulates gene expression under nitrogen deficiency. To obtain an insight into the metabolic control of this regulation, the concentrations of metabolites related to the sulfur assimilation pathway were determined. Among the metabolites, O-acetyl-l-serine (OAS), one of the precursors of cysteine biosynthesis, accumulated to higher levels under low-sulfur and high-nitrogen conditions in siliques of transgenic A. thaliana. The pattern of OAS accumulation in response to various levels of sulfur and nitrogen was similar to that of gene expression driven by the β-subunit promoter. Elevated levels of OAS accumulation were also observed in soybean cotyledons cultured under sulfur deficiency. Moreover, OAS applied to in-vitro cultures of immature soybean cotyledons under normal sulfate conditions resulted in a high accumulation of the β-subunit mRNA and protein, whereas the accumulation of glycinin was reduced. These changes were very similar to the responses observed under conditions of sulfur deficiency. Our results suggest that the level of free OAS mediates sulfur- and nitrogen-regulation of soybean seed storage-protein composition.
TL;DR: The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity.
Abstract: D-type cyclins are believed to regulate the onset of cell division upon mitogenic signaling. Here, the isolation is reported of a new D-type cyclin gene (CYCD4;1) of Arabidopsis thaliana (L.) Heynh. during a two-hybrid screen using the cyclin-dependent kinase CDC2aAt as bait. Transcription of CYCD4;1 can be induced by sucrose. The co-regulated expression of CYCD4;1 and CDC2aAt in starved suspension cultures upon mitogenic stimulation indicates that the formation of a complex between these two partners is important for the resumption of cell division activity. By in-situ hybridizations CYCD4;1 was shown to be expressed during vascular tissue development, embryogenesis, and formation of lateral root primordia. Expression during the latter process suggests that the induced expression of D-type cyclins by mitogenic stimuli might be one of the rate-limiting events for the initiation of lateral roots.
TL;DR: The results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI.
Abstract: Organellar DNA in mature pollen grains of eight angiosperm species (Actinidia deliciosa Lindl., Antirrhinum majus L., Arabidopsis thaliana (L.) Heynh., Medicago sativa L., Musa acuminata Colla, Pelargonium zonale (L.) L'Her, Petunia hybrida Vilm. and Rhododendron mucronatum (Blume) G. Don, in which the modes of organellar inheritance have been determined genetically, was observed by fluorescence microscopy using Technovit 7100 resin sections double-stained with 4',6-diamidino-2-phenylindole (DAPI) and 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)). The eight species were classified into four types, based on the presence or absence of organellar DNA in mature generative cells: namely (1) type "m+p+", which has both mitochondrial and plastid DNA (P. zonale), (2) type "m+p-", which only has mitochondrial DNA (M. acuminata), (3) type "m-p+", which only has plastid DNA (A. deliciosa, M. sativa, R. mucronatum), and (4) type "m-p-", which has neither mitochondrial nor plastid DNA (A. majus, A. thaliana, P. hybrida). This classification corresponded to the mode of organellar inheritance determined by genetic analysis. The presence or absence of mitochondrial and plastid DNA corresponded to paternal/biparental inheritance or maternal inheritance of the respective organelle, respectively. When organellar DNA was present in mature generative cells (m+ or p+), the DNA content of the organelles in the generative cells started to increase immediately after pollen mitosis one (PMI). In contrast, the DNA content of organelles in generative cells decreased rapidly after PMI when organellar DNA was absent from mature generative cells (m- or p-). These results indicate that the modes of inheritance (paternal/biparental inheritance or maternal inheritance) of mitochondria and plastids are determined independently of each other in young generative cells just after PMI.
TL;DR: The results indicate that PtdIns-(3,5)P2 plays a role in a general osmo-signalling pathway in plants.
Abstract: Cells from several different plant species synthesised a polyphosphoinositide (PPI)-like lipid when osmo-stressed. Synthesis was maximal after about 10 min and was stimulated by a variety of osmolytes. Using NaCl, the strongest response centred around 200 mM. The lipid was shown to be the novel PPI isomer phosphatidyl-inositol 3,5-bisphosphate [PtdIns-(3,5)P2] by analytical thin-layer chromatography and conversion to PtdIns(3,4,5)P3 using recombinant phosphoinositide 4-OH kinase. The results indicate that PtdIns-(3,5)P2 plays a role in a general osmo-signalling pathway in plants. Its potential role is discussed.
TL;DR: It is suggested that the coordination of expression and enzyme activities allows, in the light phase, an extensive flow of substrates into the chlorophyll-synthesizing branch of the metabolic pathway and, after the transition from light to dark, a channeling into the heme biosynthetic pathway.
Abstract: The synthesis of tetrapyrroles is regulated in anticipation of rhythmic changes in environmental conditions such as light intensity and temperature. To assess the control of the rate-limiting steps of the metabolic flow as well as the distribution of precursors for chlorophyll and heme synthesis, RNA steady-state levels and activities of enzymes involved in tetrapyrrole biosynthesis were analysed from 4-week-old tobacco (Nicotiana tobacum L.) plants grown under photoperiodically changing conditions. The kinetics of RNA levels and the enzyme activities were compared with those from plants which grew subsequent to the light/dark cycles for 48 h under constant light or dark conditions. The analysis revealed that the two peak activities for 5-aminolevulinic acid synthesis and of magnesium-protoporphyrin IX chelatase (Mg-chelatase) corresponded with the highest accumulation of the transcripts encoding glutamyl-tRNA reductase and CHL H, a subunit of Mg-chelatase, in the first half of the light period during a light/dark cycle. The activity of ferrochelatase (Fe-chelatase) and the level of its RNA showed a maximum just at the transition from light to dark and oscillated with a phase approximately opposite to that of Mg-chelatase activity. The control of 5-aminolevulinic acid synthesis and of the allocation of protoporphyrin IX to Mg- or Fe-chelatase probably reflect the functional coordination of tetrapyrrole biosynthesis in response to daily fluctuations in tetrapyrrole requirements. It is suggested that the coordination of expression and enzyme activities allows, in the light phase, an extensive flow of substrates into the chlorophyll-synthesizing branch of the metabolic pathway and, after the transition from light to dark, a channeling into the heme biosynthetic pathway. Implications for feedback control in the pathway are discussed.
TL;DR: It is proposed that this tyrosinase is specifically involved in the betalain biosynthesis of higher plants.
Abstract: A tyrosine-hydroxylating enzyme was partially purified from betacyanin-producing callus cultures of Portulaca grandiflora Hook. by using hydroxyapatite chromatography and gel filtration. It was characterized as a tyrosinase (EC 1.14.18.1 and EC 1.10.3.1) by inhibition experiments with copper-chelating agents and detection of concomitant o-diphenol oxidase activity. The tyrosinase catalysed both the formation of L-(3,4-dihydroxyphenyl)-alanine (Dopa) and cyclo-Dopa which are the pivotal precursors in betalain biosynthesis. The hydroxylating activity with a pH optimum of 5.7 was specific for L-tyrosine and exhibited reaction velocities with L-tyrosine and D-tyrosine in a ratio of 1:0.2. Other monophenolic substrates tested were not accepted. The enzyme appeared to be a monomer with an apparent molecular mass of ca. 53 kDa as estimated by gel filtration and SDS-PAGE. Some other betalain-producing plants and cell cultures were screened for tyrosinase activity; however, activities could only be detected in red callus cultures and plants of P. grandiflora as well as in plants, hairy roots and cell cultures of Beta vulgaris L. subsp. vulgaris (Garden Beet Group), showing a clear correlation between enzyme activity and betacyanin content in young B. vulgaris plants. We propose that this tyrosinase is specifically involved in the betalain biosynthesis of higher plants.
TL;DR: The data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis, and are suggested to have a role in floral organ abscission.
Abstract: Allene oxide synthase (AOS) is encoded by a single intronless gene in Arabidopsis thaliana (L.) Heynh. The promoter region of the AOS gene exhibits, in addition to the elements of a minimal promoter and the presence of general enhancers, cis-elements that, in other promoters, are responsible for stress- and ethylene-responsiveness. Arabidopsis thaliana and Nicotiana tabacum L. were transformed with a chimaeric gene consisting of a 1.9-kb 5′-upstream sequence and the first 95 nucleotides of the AOS coding sequence translationally fused to uid A encoding β-glucuronidase (GUS). Using histochemistry, GUS activity was seen in older leaves, in the bases of petioles and in stipules, during the early stages of carpel development, in maturing pollen grains and at the base of elongated filaments, as well as in abscission-zone scars. A role for jasmonates in floral organ abscission is suggested by these findings. Furthermore, the AOS promoter was activated both locally as well as systemically upon wounding. Jasmonic acid, 12-oxophytodienoic acid and coronatine strongly induced GUS activity. This induction remained confined to the treated leaf when agonists were applied locally to a leaf, suggesting that neither jasmonic acid nor 12-oxophytodienoic acid are physiologically relevant components of the systemic wound signal complex. Rather, the data show that jasmonates behave as local response regulators produced at or around the sites of action in response to appropriate triggers of their synthesis.
TL;DR: It is suggested that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response.
Abstract: The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1,5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response.
TL;DR: It is reported in this study that the extent of phosphorylation of PSII core proteins is equivalent in dimers and monomers, and directly depends on light intensity.
Abstract: The role of PSII protein phosphorylation in the oligomeric structure of the complex and in the repair of photodamaged PSII centers was studied with intact thylakoids and thylakoid membrane subfractions isolated from differentially light-treated pumpkin (Cucurbita pepo L.) leaves. A combination of sucrose gradient fractionation of thylakoid protein complexes and immunodetection with phosphothreonine and protein-specific antibodies was used. We report in this study that the extent of phosphorylation of PSII core proteins is equivalent in dimers and monomers, and directly depends on light intensity. Phosphorylated PSII monomers migrate to the stroma-exposed thylakoids, probably following damage of the D1 protein and the dissociation of the light-harvesting complex of PSII. Once in the stroma lamellae, monomers are gradually dephosphorylated to allow the reparation of the complex. First, CP43 is dephosphorylated and as a consequence of this modification it detaches from the PSII core. In addition to D1, D2 is also thereafter dephosphorylated. Phosphorylation of PSII core polypeptides probably ensures the integrity of the monomers until repair can proceed. Dephosphorylation, on the other hand, might serve the need for opening the complex and coordinating D1 proteolysis and the attachment of ribosomes.
TL;DR: There is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains.
Abstract: The composition of the two major lipidic organelles of the tapetum of Brassica napus L has been determined Elaioplasts contained numerous small (02–06 μm) lipid bodies that were largely made up of sterol esters and triacylglycerols, with monogalactosyldiacylglycerol as the major polar lipid This is the first report in any species of the presence of non-cytosolic, sterol ester-rich, lipid bodies The elaioplast lipid bodies also contained 34- and 36-kDa proteins which were shown by N-terminal sequencing to be homologous to fibrillin and other plastid lipid-associated proteins Tapetosomes contained mainly polyunsaturated triacylglycerols and associated phospholipids plus a diverse class of oleosin-like proteins The pollen coat, which is derived from tapetosomes and elaioplasts, was largely made up of sterol esters and the C-terminal domains of the oleosin-like proteins, but contained virtually no galactolipids, triacylglycerols or plastid lipid-associated proteins The sterol compositions of the elaioplast and pollen coat were almost identical, consisting of stigmasterol > campestdienol > campesterol > sitosterol ≫ cholesterol, which is consistent with the majority of the pollen coat lipids being derived from elaioplasts These data demonstrate that there is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains
TL;DR: Over 40 brassinolide like brassinosteroids (BRs) have been identified in a wide variety of plant species and like steroids such as estrogen, testosterone or ecdys one known from animals, BRs consist of a typical steroidal skeleton with specific substitutions required for biological activity.
Abstract: In 1970 a new class of putative plant growth factors was detected in pollen extracts of Brassica napus and named "brassins" (Mitchell et al. 1970). Brassinolide (Fig. 1), a novel steroid of unique structure, was shown to be responsible for the growth-promoting activity of "bras sins" (Grove et al. 1979). Today over 40 brassinolide like brassinosteroids (BRs) have been identified in a wide variety of plant species (reviewed in: Mandava 1988; Marquardt and Adam 1991; Adam et al. 1996; Sasse 1997). Like steroids such as estrogen, testosterone or ecdys one known from animals, BRs consist of a typical steroidal skeleton with specific substitutions required for biological activity. Several bioassays, including the bean second-internode bioassay (Mitchell and Livingston 1968), the Raphanus sativus test (Takasuto et al. 1983), the tomato test (Takasuto et al. 1983) and the rice lamina inclination test (Wada et al. 1981; Arima et al. 1984), have been used to determine the bioactivity of either naturally occurring substances isolated from plant tissues or chemically synthesised analogs. The most active BRs exhibit either a 6-oxo function (as in the case of castasterone) or a lactone structure (as in the case of brassinolide) at the B ring which in the latter case is seven-numbered. Furthermore, most of the naturally occurring BRs thus far identified are hydroxylated at the C-2, C-3, C-22 and C-23 positions, the latter two being in R configuration. Structural variations other than the substitution patterns of rings A and B lie in the different alkylations of C-24. The most highly active BRs
TL;DR: Electron-microscopy analysis showed dramatic alterations both to the organization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls, and DP administration inhibited cell cycle progression.
Abstract: Post-translational hydroxylation of peptide- bound proline residues, catalyzed by peptidyl-prolyl-4 hydroxylase (EC 1.14.11.2) using ascorbate as co- substrate, is a key event in the maturation of a number of cell wall-associated hydroxyproline-rich glycoproteins (HRGPs), including extensins and arabinogalactan- proteins, which are involved in the processes of wall stiAening, signalling and cell proliferation. Allium cepa L. roots treated with 3,4-DL-dehydroproline (DP), a specific inhibitor of peptidyl-prolyl hydroxylase, showed a 56% decrease in the hydroxyproline content of HRGP. Administration of DP strongly aAected the organization of specialized zones of root development, with a marked reduction of the post-mitotic isodiametric growth zone, early extension of cells leaving the meristematic zone and a huge increase in cell size. Electron-microscopy analysis showed dramatic alterations both to the orga- nization of newly formed cell walls and to the adhesion of the plasma membranes to the cell walls. Moreover, DP administration inhibited cell cycle progression. Root tips grown in the presence of DP also showed an increase both in ascorbate content (+53%) and ascor- bate-specific peroxidase activity in the cytosol (+72%), and a decrease in extracellular ''secretory'' peroxidase activity ()73%). The possible interaction between HRGPs and the ascorbate system in the regulation of both cell division and extension is discussed.
TL;DR: Two closely related 12-oxophytodienoic acid reductases, OPR1 and OPR2, were identified on a 7079-bp-long genomic fragment from Arabidopsis thaliana and revealed differences in OPR-promoter-driven GUS expression in flowers.
Abstract: The genes of two closely related 12-oxophytodienoic acid reductases (EC 1.3.1.42), OPR1 and OPR2, were identified on a 7079-bp-long genomic fragment from Arabidopsis thaliana (L.) Heynh. The organization of these two genes was determined and putative cis elements were identified. Promoter-beta-glucuronidase (GUS) fusions expressed in transgenic Arabidopsis thaliana and Nicotiana tabacum L. plants revealed differences in OPR-promoter-driven GUS expression in flowers. While the OPR1 promoter directed GUS expression in young seeds, the OPR2 promoter directed pollen-specific expression. Both OPR1 and OPR2, were predominantly expressed in roots. Stress treatments, like local and systemic wounding, UV-C illumination and coldness, resulted in transient changes in steady-state OPR mRNA levels, but no concurrent changes in polypeptide level or enzyme activity were detected.
TL;DR: Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation, suggesting Brassica napus oilseed may provide a better system for PHB production.
Abstract: Polyhydroxyalkanoates (PHAs) comprise a class of biodegradable polymers which offer an environmentally sustainable alternative to petroleum-based plastics. Production of PHAs in plants is attractive since current fermentation technology is prohibitively expensive. The PHA homopolymer poly(beta-hydroxybutyrate) (PHB) has previously been produced in leaves of Arabidopsis thaliana (Nawrath et al., 1994, Proc Natl Acad Sci USA 91: 12760-12764). However, Brassica napus oilseed may provide a better system for PHB production because acetyl-CoA, the substrate required in the first step of PHB biosynthesis, is prevalent during fatty acid biosynthesis. Three enzymatic activities are needed to synthesize PHB: a beta-ketothiolase, an acetoacetyl-CoA reductase and a PHB synthase. Genes from the bacterium Ralstonia eutropha encoding these enzymes were independently engineered behind the seed-specific Lesquerella fendleri oleate 12-hydroxylase promoter in a modular fashion. The gene cassettes were sequentially transferred into a single, multi-gene vector which was used to transform B. napus. Poly(beta-hydroxybutyrate) accumulated in leukoplasts to levels as high as 7.7% fresh seed weight of mature seeds. Electron-microscopy analyses indicated that leukoplasts from these plants were distorted, yet intact, and appeared to expand in response to polymer accumulation.